J Integr Plant Biol. ›› 2001, Vol. 43 ›› Issue (1): 24-28.

• Research Articles • Previous Articles     Next Articles

Changes in Inhibitory Effect of Vanadate on the Plasma Membrane H+-ATPase Under Trypsin Treatment

QIU Quan-Sheng, ZHANG Nan   

Abstract:

The changes in inhibitory effect of vanadate on the plasma membrane H + ATPase were studied with mild trypsin treatment using plasma membrane vesicles purified from soybean (Glycine max L.)hypocotyles by sucrose gradient centrifugation. Results showed that under mild trypsin treatment the ATPase ATP hydrolysis activity was increased significantly. It was also found that the inhibitory effect of vandate was reduced after proteolysis. In the presence of 2 mmol/L vanadate, the ATP hydrolysis activity of the cleaved ATPase was inhibited by only 53.49%,while that of the un-cleaved ATPase was inhibited by 64.13%. Kinetic studies indicated that both the Km values and the inhibition type of vanadate were not affected by trypsin treatment. Upon proteolysis, Km remained as 0.34 mmol/L,while vanadate was still an uncompetitive inhibitor. Taking together, the structure and activity of the ATPase phosphatase domain were affected by trypsin treatment, implying that this domain might be regulated by the C-terminal end of the plasma membrane H+ ATPase.

胰蛋白酶处理对质膜H+-ATPase钒酸钠抑制效应的影响
邱全胜* 张楠
(北京师范大学生命科学学院,北京100875)

采用经蔗糖密度梯度法纯化的大豆(Glycine max L.)下胚轴质膜微囊为材料,分析了胰蛋白酶处理对质膜H+-ATPase钒酸钠抑制效应的影响。实验结果显示,温和胰蛋白酶处理显著提高H+-ATPase的ATP水解活力。并且发现酶切处理降低了钒酸钠对ATPase 的抑制效应,当钒酸钠浓度为2 mmol/L时,ATPase活力仅被抑制53.49%,而未经酶切的对照组则被抑制64.13%。ATP水解动力学分析表明,胰蛋白酶酶切处理既不影响ATP水解的Km值也不影响钒酸钠的抑制类型,酶切前后的Km值都等于0.34 mmol/L,并且都属于反竞争抑制。以上结果显示胰蛋白酶酶切处理可能改变了磷酸酶结构域的结构而影响了钒酸钠的抑制效应,暗示C-末端调节着磷酸酶结构域的结构和功能。

关键词: 大豆下胚轴;质膜H+-ATPase C-末端自抑制结构域;磷酸酶结构域;钒酸钠;胰蛋白酶

Key words: soybean hypocotyles, plasma membrane H+ ATPase, C-terminal autoinhibitory domain, phosphatase domain, vanadate, trypsin

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